Drosophila pseudoobscura Library Construction
The strain chosen for this project is inbred line MV2-25 isolated by Dr. Wyatt Anderson of the University of Georgia from flies collected at Mesa Verde, Colorado in 1996. Eight iso-female lines from this Mesa Verde collection were used to establish a population cage on 7/8/97. After approximately 50 generations, 25 inbred lines were set up using virgin males and females from a single pair of parents. Every generation one brother and sister (all virgins) were collected and crossed to each other. After 16 generations of inbreeding, MV2-25 was chosen based on its robust growth. Dr. Anderson confirmed the species identity cytologically by inspection of chromosomal banding patterns. The line was expanded and embryos collected for genomic DNA preparation by Dr. Stephen Richards at the BCM-HGSC. We would like to thank Dr. Anderson and Dr. Yong-Kyu Kim for their help with this strain. The strain is available from the Tucson Drosophila species stock center.
2 kb Plasmid Library
High molecular weight DNA was isolated and randomly sheared to produce fragments 1.8 - 2.2 kb and 2.5 - 3.0 kb in length. After end-repair and adaptor ligation, inserts were annealed with complementary adaptor-modified pUC18 and used to transform XL-10 Gold competent cells essentially as described in Andersson, et al., (1996), Analytical Biochemistry 236: 107-113.
10 kb Plasmid Library
High molecular weight DNA was isolated and randomly sheared to produce fragments 9-10 and 10-11 kb in length. After end-repair and adaptor ligation, inserts were annealed with complementary adaptor-modified pBR322 to produce a low-medium copy library in XL-10 Gold.
The BCM-HGSC libraries are archived but are not yet available for distribution.
BAC and Fosmid Libraries
These libraries were constructed and are available from Pieter DeJong's laboratory (BACPAC) at the Children's Hospital in Oakland, CA.
A D. pseudoobscura embyonic cDNA library was made available to us by Mark Stapleton and Gerry Rubin at the BDGP. The library was prepared by Ling Hong into pOTB7. It will be used for EST sequencing and some full length cDNA sequencing.